Technical Abstract:
The presence of strong biofilm forming microflora could potentially enhance the survival of Escherichia coli O157:H7 (EcO157) in harsh environment. One strain of Ralstonia insidiosa isolated from produce processing environments, previously displayed a synergistic interaction with EcO157 in dual-species biofilms, as indicated by the increased biomass. The objectives of this study were to examine the possibility of the occurrence under different mimicking environmental conditions and elucidate the potential parameter that might affect this interaction. The existence of EcO157 in biofilms was examined using tissue culture plates in the presence or absence of R. insidiosa. Three types of nutrient source, two temperatures, and two culture methods were selected to elucidate the consistency of the synergism. Cells in biofilms were enumerated by plating and biofilm structure was examined using Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). Three replicates were conducted for each study. Co-culture significantly increased (P<0.05) the incorporation of EcO157 in dual-species biofilms in all tested conditions. Within 4 hrs of inoculation, the presence of pre-formed R. insidiosa biofilms significantly enhanced the attachment of EcO157 for 0.83 log CFU/cm2 (P<0.05) compared to that on a plain plate. This enhanced initial attachment of EcO157 was not observed when it was co-inoculated with R. insidiosa, indicating the importance of R. insidiosa biofilm in this interaction. Result aslo showed that the enhanced incorporation of EcO157 required living R. insidiosa rather than the biomass produced by R. insidiosa in a dynamic culture system. CLSM microscopic examination of dual-species biofilms revealed a unique arrangement of the two strains, where EcO157 microcolones were often encapsulated by R. insidiosa on the solid substrate. Additionally, SEM microscope images displayed heterogeneous biofilms with well-structured extracellular polysaccharides that supporting and connecting bacteria cells in dual-species biofilms. This study provided evidence that biofilm formation by native microflora facilitates the survival of foodborne pathogens such as EcO157 through a synergistic interaction.